PROJECT SUMMARY/ABSTRACT Buprenorphine (BUP) is an effective treatment for opioid use disorder (OUD); however, treatment with BUP during pregnancy is associated with a high rate of physical dependence in offspring and leads to a withdrawal syndrome known as neonatal abstinence syndrome (NAS). Arguably, BUP is currently the best treatment for OUD during pregnancy because it improves maternal?child outcomes relative to other or no treatments, but the high rate of NAS associated with BUP indicates that improved treatments are needed. Understanding the mechanisms that promote BUP-associated NAS can facilitate the development of improved treatments. Evidence suggests that fetal exposure to a major active metabolite of BUP, called norbuprenorphine (NorBUP), may promote BUP-associated NAS. Moreover, NorBUP does not appear to contribute to the therapeutic effects of BUP, indicating that NorBUP is not vital for BUP treatment. Therefore, reducing or eliminating NorBUP formation is a potential strategy to improve BUP treatment. However, the degree to which NorBUP contributes to NAS is currently undefined because there have been no means to distinguish the effects of BUP and NorBUP following BUP administration. This proposal seeks to develop a new tool, deuterated buprenorphine (BUP-D2), that will distinguish the contributions of NorBUP and BUP in BUP-associated NAS. Preliminary data suggests that BUP-D2 resists metabolism to NorBUP relative to BUP but fully retains the opioid activity of BUP. These properties will elucidate whether fetal exposure to NorBUP substantially contributes to BUP-associated NAS. The experiments proposed in this study will determine whether fetal exposure to NorBUP is reduced following chronic prenatal treatment with BUP-D2 relative to BUP, and will characterize opioid receptor affinity and activity of BUP-D2. We hypothesize that relative to BUP, BUP-D2 will decrease fetal exposure to NorBUP when administered during pregnancy and will retain the opioid activity of BUP. Aim 1 will compare concentrations of NorBUP in fetal brains harvested during late gestation following chronic prenatal treatment with BUP-D2 versus BUP. Aim 1 also will measure concentrations of parent drugs, NorBUP, and other major metabolites in maternal plasma, maternal brain, fetal plasma, and fetal brain to determine if compensatory pharmacokinetics changes occur with these compounds following BUP-D2 administration. Aim 2 will characterize the affinity and activity of BUP-D2 versus BUP with in vitro assays using CHO cells transfected with human mu, delta, or kappa opioid receptors. If BUP- D2 reduces fetal brain exposure to NorBUP and retains the activity of BUP, it will be used in future studies to determine the impact of fetal exposure to NorBUP during prenatal BUP treatment. If NorBUP is confirmed to exert adverse effects on the fetus, BUP-D2 will be investigated as a potential improved therapy for OUD during pregnancy.